CN101605890A - The novel promoter nucleic acid molecule of derived from corynebacterium glutamicum, comprise described promotor recombinant vectors, comprise the host cell of described recombinant vectors and utilize the method for described host cell expression gene - Google Patents
The novel promoter nucleic acid molecule of derived from corynebacterium glutamicum, comprise described promotor recombinant vectors, comprise the host cell of described recombinant vectors and utilize the method for described host cell expression gene Download PDFInfo
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Abstract
The invention provides the novel promoter nucleic acid molecule of the nucleotide sequence of SEQ ID NO:1 with derived from corynebacterium glutamicum or 2, the recombinant vectors that comprises described promotor is with described carrier transformed host cells and the method for utilizing described host cell expression goal gene.
Description
Technical field
The present invention relates to derived from corynebacterium glutamicum (Corynebacterium glutamicum) novel promoter nucleic acid molecule, comprise the recombinant vectors of described promotor, with described recombinant vectors transformed host cells with utilize the method for described host cell expression target gene.
Background technology
Bar shaped bacteria has been widely used in the chemical substance that has multiple application in the industry such as being created on animal-feed, pharmacy, food, comprises L-Methionin, L-Threonine and multiple nucleic acid.In order to utilize genetic engineering and metabolic engineering technology, need selectivity to regulate the expression of gene that relates in the multiple pathways metabolism in the bar shaped bacteria, and need to be effective to the promotor of these generegulation thus by described bar shaped bacteria exploitation high yield bacterial strain.
The ordinary method of separating promotor comprises: (1) uses promoter probe vector to clone the method for the genomic DNA fragment of the reporter gene upstream of only expressing at random when clone's fragment comprises promoter activity; (2) utilize the method for hybrid method based on gene-specific probe isolated genes and promotor thereof from gene library; (3) utilize the gene library differential of derivable cDNA probe and non--derivable cDNA probe to hybridize.
In the genetic expression in bar shaped bacteria, gene expression under the control of their original promotors usually (Vasicova, P., etc., bacteriology magazine (J.Bacteriol.), 181,6188-6191, (1999), etc.).Yet with other industrial microorganisms such as intestinal bacteria (Escherichia coli), subtilis (Bacillus subtilis) etc. are different, and the typical structure that is used at the promoter sequence of bar shaped bacteria genetic expression still is unknown.Therefore, the promotor that is used for bar shaped bacteria by following steps exploitations: from to eliminating promoter region the relevant gene of the resistance of microbiotic such as paraxin, utilize suitable restrictive diges-tion to introduce isolating chromosomal dna fragment from bar shaped bacteria, use dna molecular conversion bar shaped bacteria that generates thus and the antibiotics resistance (Eikmanns that assesses the bacterial strain that obtains to this promotor site, B.J., Deng, gene (Gene), 102,93-98, (1991); Patek, M., etc., microbiology (Microbiology), 142,1297-1309, (1996)).Yet the promoter sequence of conventional exploitation still need improve at the selectivity of genetic expression, gene expression efficiency etc.
We develop the novel promoter nucleic acid molecule of derived from corynebacterium glutamicum through the following steps: search and the promoter region of inferring by polymerase chain reaction (PCR) amplification, described promotor of inferring is incorporated into the initiation site of the lysC gene that lacks promotor and determines that by the generation of Methionin thereby the active variation of lysC selects effective promotor.
Summary of the invention
Technical problem
The invention provides the novel promoter nucleic acid molecule of derived from corynebacterium glutamicum.
The present invention also provides the recombinant vectors of the novel promoter nucleic acid molecule that comprises derived from corynebacterium glutamicum.
The present invention also provides the recombinant vectors transformed host cells with the novel promoter nucleic acid molecule that comprises derived from corynebacterium glutamicum.
The present invention also provides the method for expressing target gene with the recombinant vectors transformed host cells of the novel promoter nucleic acid molecule that comprises derived from corynebacterium glutamicum of utilizing.
Technical scheme
According to an aspect of of the present present invention, provide promoter nucleic acid molecule with SEQ ID NO:1 or 2 nucleotide sequences.
Nucleotide sequence according to promoter nucleic acid molecule of the present invention can be modified to a certain degree by one of technology such as the orthogenesis of some recent development or mutagenesis of site-guidance.Those technician in this area should understand easily with promoter sequence of the present invention have 70% or the nucleotide sequence of higher homology be equivalent to promotor of the present invention, condition is its promoter activity that keeps expressing target gene.
Therefore, can comprise that according to promoter nucleic acid molecule of the present invention nucleotide sequence with SEQ ID NO:1 or 2 has 70% or higher homology and can be used as the nucleotide sequence that promotor is used.
Term " homology " is used for representing the degree identical with the sequence of wild-type nucleic acid sequence herein.Promotor of the present invention can comprise the promotor with such dna sequence dna, the nucleotide sequence 75% of described dna sequence dna and novel promotor of the present invention or higher, preferred 85% or higher, more preferably 90% or higher with most preferably 95% or more the highland is identical.Homology can be purchased software by naked eyes or use and compare.According to being purchased software program, can calculating homology between the two or more sequences with percentage ratio (%), and can calculate the homology (%) between the flanking sequence.
In addition, promoter nucleic acid molecule of the present invention can comprise the promoter nucleic acid molecule of derived from corynebacterium glutamicum, and it is selected from by comprising and molecular group of the startup of above-mentioned nucleotide sequence complementary nucleotide sequence.
Term " complementation " is used for herein, between expression Nucleotide or the nucleic acid, for example, between two chains of double chain DNA molecule or Oligonucleolide primers and wait possible hybridization or base pairing between the primer binding site of the single-chain nucleic acid template that checks order or increase.
In addition, the promoter nucleic acid molecule of derived from corynebacterium glutamicum of the present invention comprises the function equivalent of the promoter nucleic acid molecule of derived from corynebacterium glutamicum.The function equivalent that comprises the promotor of the present invention of its function fragment can comprise the variant with at least one base replacement, disappearance, insertion or its combination.
Corynebacterium glutamicum promoter nucleic acid molecule of the present invention is the promotor that is derived from bar shaped bacteria, and preferably has the effectiveness work at prokaryotic cell prokaryocyte, expresses the promotor of goal gene particularly in intestinal bacteria and the bar shaped bacteria.
Term " promotor " is used for herein, thereby the expression RNA polymerase combines the DNA zone that initial gene is transcribed with it, and it is positioned at the upstream of mRNA transcription initiation site, to 5 ' direction.
Promotor of the present invention with SEQ ID NO:1 of the present invention or 2 nucleotide sequences can be the promotor of gene NCgl1504 (SEQ ID NO:11) and gene NCgl1305 (SEQ ID NO:12), and described gene is to select by the gene expression amount of about 3000 genes of analyzing Corynebacterium glutamicum ATCC13032.
In this article, " gene with nucleotide sequence NCgl1504 " and " gene with nucleotide sequence NCgl1305 " refers to that not only derived from corynebacterium glutamicum ATCC 13032 has the gene of SEQ IDNOS:11 and 12 nucleotide sequences respectively, also refers to express in belonging to the microorganism that excellent bacillus (Corynebacterium) belongs to the gene with those essentially identical products of being expressed by NCgl1504 or NCgl1305.Term " NCgl1504 gene " and " NCgl1305 gene " refer to " having the gene of nucleotide sequence NCgl1504 " and " gene with nucleotide sequence NCgl1305 " respectively.Term " basic identical " is used for herein, expression activity and regulation mechanism.Gene with nucleotide sequence NCgl1504 can be the gene with nucleotide sequence of SEQ ID NOS:11, and the gene with nucleotide sequence NCgl1305 can be the gene with nucleotide sequence of SEQ ID NO:12.
Can utilize standard molecular biological technique according to promoter nucleic acid molecule of the present invention, for example separate or preparation by the PCR that uses suitable primer sequence.It can also be by using the standard synthetic technology preparation of automatization DNA synthesizer.
The present invention is the promotor by comprising the nucleotide sequence with SEQ ID NO:1 or 2 and the recombinant vectors of the encoding sequence of the target gene that is connected with described promotor operability also.
Term " carrier " is used for herein, and expression comprises the DNA construct of such dna sequence dna, and described dna sequence dna is connected be applicable to the control sequence operability of expressing in proper host cell.Described suitable control sequence comprise instruct the promotor transcribe, regulate described any operator gene sequence of transcribing, the sequence of mRNA ribosome bind site that coding is suitable and be used to the sequence transcribing and translate.Described carrier can be plasmid, bacteriophage particles or only be potential genome inset.When carrier transformed compatible host, this carrier can be independent of host genome and duplicate and work, or can be incorporated in some cases in host's the genome.Term " operability connects " is used for herein, represents that gene to be expressed is connected with its control sequence is functional, so that this gene is correctly expressed.
Comprise that the recombinant vectors according to Corynebacterium glutamicum promoter nucleic acid molecule of the present invention can be connected with the gene operability of coding multiple proteins, thereby be recombinantly produced target protein.It can be the lysC of coding E.C. 2.7.2.4., coding dihydrodipicolinate reductase's dapB etc. that desire is used the target gene of vector expression of the present invention, but is not limited in this.
According to target gene of the present invention can be the lysC of coding E.C. 2.7.2.4..The lysC of coding E.C. 2.7.2.4. can have base sequence (Ikeda etc., microbiology and biotechnology applications (Appl Microbiol Biotechnol.) in the February, 2002 of SEQ ID NO:13; 58 (2): 217-23 uses Corynebacterium glutamicum gene group information to produce the neomethodology (A novelmethodology employing Corynebacterium glutamicum genome information togenerate a new L-lysine-producing mutant) of novel L-Methionin-generation mutant).
According to recombinant vectors of the present invention can be pDZ-NCgl1504-lysC, and it comprises the promotor of the nucleotide sequence with the SEQ ID NO:1 that is connected with lysC encoding sequence (SEQ ID NO:13) operability as target gene.Described recombinant vectors can also be pDZ-NCgl1305-lysC, and it comprises the promotor of the nucleotide sequence with the SEQ ID NO:2 that is connected with lysC encoding sequence (SEQ ID NO:13) operability as target gene.
The present invention also provides and uses the recombinant vectors transformed host cells, and described recombinant vectors comprises the promotor of the nucleotide sequence with SEQID NO:1 or 2.
Described host cell, for example, prokaryotic cell prokaryocyte, preferred intestinal bacteria and bar shaped bacteria, more preferably the bar shaped bacteria recombinant vectors that can be produced transforms, so that Corynebacterium glutamicum promoter nucleic acid molecule is connected with the gene operability of the target protein of encoding, thereby expresses this target protein.
Described " bar shaped bacteria " can be to belong to the bacterium that Corynebacterium or tyrothricin (Brevibacterium) belong to, particularly, and Corynebacterium glutamicum and more specifically, Corynebacterium glutamicum ATCC 13032.Bar shaped bacteria of the present invention can comprise other bacterial strains of Corynebacterium, Corynebacterium thermoaminogenesFERM BP-1539, brevibacterium flavum (Brevibacterium flavum) ATCC 14067, brevibacterium (Brevibacterium lactofermentum) ATCC 13869 and generation amino acid whose mutant of L-or Corynebacterium glutamicum KFCC 10881, Corynebacterium glutamicum KFCC 11001 etc.
Term " conversion " is used for herein, and expression is introduced DNA among the host by this way, and described mode is that it can duplicate as extra-chromosomal element or by chromosomal integration.
Host cell can be the excellent bacillus that transforms with such recombinant vectors, and described recombinant vectors comprises the promotor and the lysC that is connected with described promotor operability (SEQ ID NO:13) of the nucleotide sequence with SEQ ID NO:1.Host cell is Corynebacterium glutamicum (preserving number KCCM10831) preferably.
Host cell can also be the excellent bacillus that transforms with such recombinant vectors, and described recombinant vectors comprises the promotor of the nucleotide sequence with SEQ ID NO:2 and the lysC that is operatively connected with described promotor (SEQ ID NO:13).Host cell is Corynebacterium glutamicum (preserving number KCCM10830) preferably.
The present invention also provides the method for expressing target gene, and described method comprises cultivates the recombinant vectors transformed host cells that apparatus has the promoter nucleic acid molecule that is derived from Corynebacterium glutamicum.
With the target gene that the promotor operability of the nucleotide sequence with SEQ ID NO:1 or 2 connects can be the coding proteinic gene relevant with Threonine with synthetic end product such as Methionin.Term " expression target gene " is used for herein, and expression generates the end product of route of synthesis I, and described route of synthesis I relates to by target gene encoded protein matter.Therefore, the method for expressing target gene can be by cultivating with comprising that the recombinant vectors host transformed of described target gene generates the method for the end product of route of synthesis, relating in described route of synthesis by described target gene encoded protein matter.
Described final product can be a Methionin.Be that the present invention can provide the method that generates Methionin, it comprises cultivation by the recombinant vectors transformed host cells, described recombinant vectors comprise coding Methionin synthetic in the promotor of proteinic lysC that relates to and the nucleotide sequence that is connected with this gene operability with SEQ ID NO:1 or 2.
In according to Methionin synthetic method of the present invention, described host cell can be Corynebacterium glutamicum KCCM 10831 or bar shaped bacteria (Coryneform bacterium) KCCM10830 that is transformed by recombinant vectors, described recombinant vectors comprises and target gene, i.e. the promotor of the nucleotide sequence with SEQ ID NO:1 or 2 of the lysC operability of SEQ ID NO:13 connection.
The cultivation of transformed host cells (transformant) can be carried out according to method commonly used in this area.Known cultural method is recorded and narrated at Chmiel, (Bioprozesstechnik 1.Einfuhrung in dieBioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991); And among the Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
The substratum that is used for this cultivation need reach the requirement of the specific bacterial strain of growing in a suitable manner.The substratum that is used for coryneform bacterial strains is disclosed in, for example, conventional bacteriological method handbook (Manual of Methods forGeneral Bacteriology), U.S. bacteriology association (American Society for Bacteriology). Washington, the U.S. is in 1981.The carbon source of substratum can be carbohydrate such as glucose, sucrose, lactose, fructose, maltose, starch and Mierocrystalline cellulose, oil ﹠ fat such as soya-bean oil, heliotrope oil, castor-oil plant are by (casteroil) and cocounut oil, lipid acid such as palmitinic acid, stearic acid and linolenic acid, alcohol such as glycerine and ethanol and organic acid are such as acetate.Carbon source can be used alone or as a mixture.Nitrogenous source also can be peptone, yeast extract, meat extract, malt extract, corn steep liquor, soyflour and urea, or mineral compound, for example, and ammonium sulfate, ammonium sulfide, ammonium phosphate, volatile salt and ammonium nitrate.Nitrogenous source can be used alone or as a mixture.The phosphorus source can be potassium primary phosphate, dipotassium hydrogen phosphate or its sodium salt.In addition, described substratum should comprise the necessary metal-salt of growth, such as sal epsom or ferric sulfate.At last, described substratum can also comprise the essential material of growth, such as amino acid and VITAMIN.In addition, can also in substratum, add suitable precursor.Those compositions of substratum are can be in culturing process a collection of or based on joining in the substratum of continuing.
The pH of substratum can use basic cpd such as sodium hydroxide, potassium hydroxide and ammoniacal liquor, or acidic cpd such as phosphoric acid or sulfuric acid, regulates by rights.In addition, utilize antifoams such as fatty acid polyglycol ester can prevent that foam from forming.Oxygen or contain-carrier of oxygen, for example air can be incorporated in the substratum, thereby keeps inflated condition.The temperature of substratum can be in the scope of 20-45 ℃ and preferred 25-40 ℃.The target amount of substance that continues to cultivate until generating reaches its maximum value.Usually, cultivation was carried out 10-160 hour.
To the present invention be described in more detail with reference to the following example.The following example only is an illustrative purposes for example, and is not intended to limit scope of the present invention.
Advantageous effects
According to the present invention, the novel nucleic acid sequence of promoter that derived from corynebacterium glutamicum is provided is to be used for the effective expression goal gene.
Description of drawings
Above and other purpose of the present invention, feature and other advantages will obtain by the detailed description below in conjunction with accompanying drawing more clearly understanding, in described accompanying drawing:
Fig. 1 schematically shows according to embodiment of the present invention, in order to study the process that promoter activity prepares carrier.
The invention pattern
Embodiment 1: preparation comprises the recombinant vectors of novel promoter sequence
1. select candidate gene about the novel promotor of derived from corynebacterium glutamicum
In the 5L fermentation container, cultivate Corynebacterium glutamicum ATCC 13032, and collecting cell.Utilize genomic dna chip (cDNA chip, Genomictree, company, Korea S) to determine the mRNA expression level of about 3000 kinds of genes of Corynebacterium glutamicum ATCC13032.To account for the candidate gene that 0.88% and 0.43% NCgl1504 and NCgl1305 gene are elected to be the novel promotor of the present invention respectively based on total genomic expression.
2. the dna fragmentation of the amplification promoter region of inferring
The nucleotide sequence of Corynebacterium glutamicum gene group is to have determined fully and known (microbiology and biotechnology applications (Appl.Microbiol.Biotechnol.), 62 (2-3), 99-109 (2003): GenBank registration number NC_003450).The sequence information of protein (NCgl1504 and NCgl1305) is available from the Genbank of national health institute (NIH) (U.S.) database.Be positioned at the promoter region of the inferring (promoter region of SEQ ID NO:1-NCgl1504 of range gene open reading frame (ORF) upstream in order to increase, the promoter region of SEQID NO:2-NCgl1305), based on the synthetic primer 1-4 that comprises EcoRV/BamHI and NdeI restriction site of the nucleotide sequence of report.In PCR, the chromosomal DNA that utilizes Corynebacterium glutamicum ATCC 13032 is as template, use primer 1 and 2 (SEQ ID NOS:3 and 4) and primer 3 and 4 (SEQ ID NOS:5 and 6) respectively, with 30 94 ℃ of sex change, 55 ℃ of annealing 1 minute and 0 second circulation of 72 ℃ of polyase 13s, promoter region [the Sambrook etc. that infer of amplification NCgl1504 and NCgl1305 gene, molecular cloning, laboratory manual (Molecular Cloning, a LaboratoryManual) (1989), cold spring harbor laboratory (Cold Spring Harbor Laboratories)].
3. thereby preparation is used for the recombinant vectors that chromosomal integration is determined promoter activity
The promoter region of inferring activity in excellent bacillus karyomit(e) for definite NCgl1504 that obtains and NCgl1305, we use carrier pDZ to carry out chromosomal integration (Korean Patent Application No. 1-2006-089672), it is to utilize pACYC177 (New England's biology laboratory (New England Biolab) by Cheiljedang Co., Ltd., GenBank accetion#X06402), promptly a kind of colibacillary cloning vector is developed as underlying carrier.For gene being inserted in the excellent bacillus karyomit(e), before the novel promotor that will have a nucleotide sequence of SEQ ID NOS:1 or 2 is inserted into the lysC initiation site, thereby obtain pDZ-Ncgl1504-lysC or pDZ-Ncgl1305-lysC carrier respectively.
Prepare recombinant vectors in the following manner, wherein the promotor site of inferring with NCgl1504 and NCgl1305 is inserted in the lysC gene.LysC for the SEQ ID NO:13 that increases, the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 is used as template, and utilize primer 5 and 6 (SEQ ID NOS:7 and 8) and primer 7 and 8 (SEQ ID NOS:9 and 10) to carry out PCR (PCR condition: 30 94 ℃ of sex change, 30 seconds circulations of 55 ℃ of annealing, 1 minute and 72 ℃ extension).With the LysC fragment of EcoRV and Klenau digestibility and utilization TOPO clone's test kit (Invitrogen) amplification, these two blunt-ended fragments connect, and are cloned among the pCR2.1-lysC.Then, utilize the promotor site of EcoRV and NdeI cracking pCR.2.1-lysC and NCgl1504 or NCgl1305, and utilize dna ligase that the novel promotor of SEQ ID NO:1 or 2 is inserted into this restriction site.Then, clone's fragment is transferred in the pDZ carrier, thus preparation pDZ-Ncgl1504-lysC and pDZ-Ncgl1305-lysC carrier, as shown in fig. 1.
Embodiment 2. usefulness comprise that the recombinant bacterial strain of novel promoter sequence transforms
As disclosed ground among microbiology and biotechnology applications (Appl.Microbiol.Biotechnol.) (1999) 52:541-545, utilize the recombinant vectors pDZ-NCgl1504-lysC or the pDZ-NCgl1305-lysC of preparation, transform Corynebacterium glutamicum KFCC 10881, i.e. L-Methionin-generation bacterial strain by electricimpulse.Comprising 25mg/L kantlex (10g/L beef extract, the 10g/L peptone, the 5g/L yeast extract, 5g/L sodium-chlor, 3.7g/L brain heart infusion (BHI) and 9.1g/L Sorbitol Powder) selective medium in the bacterial strain that select to transform, wherein the novel promotor on the reactor is incorporated in the karyomit(e) by homologous recombination.Whether in the solid medium that comprises X-gal (5-bromo-4-chloro-3-indyl-β-D-galactoside), become blue according to this bacterial strain, can identify the insertion of carrier.In nutritional medium, by shaking, cultivated such bacterial strain 8 hours at 30 ℃, carrier is inserted in the karyomit(e) by intersecting for the first time in described bacterial strain, dilutes it to 10 respectively
-4-10
-10And be applied on the solid medium that comprises the X-semi-lactosi.Most of bacterium colonies demonstrate blueness, and therein by intersect for the second time remove the carrier sequence of inserting bacterial strain by selecting the white colony screening.Utilization is at the susceptibility of kantlex, identifies and confirms selected bacterium colony fully by order-checking.The promotor of lysC gene wherein is called CA01-0037 by the displaced bacterial strain of Ncgl1504, and wherein the promotor of lysC gene is called CA01-0036 by the displaced bacterial strain of Ncgl1305, and they were on December 28th, 2006, were deposited in Korea S microorganism culturing center (Korean Culture Center of Microorganisms) with KCCM 10831 and KCCM 10830 respectively.
Embodiment 3: the activity of promoter sequence in the excellent bacillus
Cultivate the bacterial strain that transforms, thereby press the activity of following analysis promoter sequence.
With 1: 20 ratio, the Corynebacterium glutamicum strain of various conversions is inoculated into 25ml substratum [20g glucose, 5g ammonium sulfate, 5g yeast extract, 1.5g urea, 4g KH are housed
2PO
4, 8g K
2HPO
4, 0.5g MgSO
47H
2O, 150 μ g vitamin Hs, 1.5mg vitamin, the 3mg calcium pantothenate, 3mg niacinamide (based on 1L distilled water), pH 7.2] the shaking in the bottle of 250ml belt edge-baffle plate (corner-baffle), and at 30 ℃, cultivation reaches logarithmic growth (OD in mid-term up to this culture when shaking with 200rpm
600=10).When stopping this cultivation, by centrifugal collecting cell, and be suspended in the 100mM Tris-HCl damping fluid (pH 7.0), by ultrasonic treatment, and carry out high speed centrifugation then, to obtain supernatant liquor.The activity (Black and Wright (1955b)) of lysC enzyme is measured in use from the 1mg protein of this supernatant liquor.As a result, the promotor about lysC has been confirmed the active change of lysC in NCgl1504 or the displaced bacterial strain of NCgl1305 therein, as shown in following table.
Table. promoter activity is compared in the interpolation by Methionin
[table 1]
[table]
| Bacterial strain | The relative extent of promoter expression |
| ??KFCC?10881 | ??1 |
| ??CA01-0036(KCCM?10830) | ??0.74 |
| ??CA01-0037(KCCM?10831) | ??1.67 |
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Sequence table
＜110〉CJ Co., Ltd.
＜120〉the novel promoter nucleic acid molecule of derived from corynebacterium glutamicum
<160>13
<170>KopatentIn?1.71
<210>1
<211>500
<212>DNA
＜213〉Corynebacterium glutamicum (Corynebacterium glutamicum)
<220>
＜221〉promotor
<222>(1)..(500)
＜223〉NCgl1504 promotor
<400>1
agcgttgccc?ctaaagggct?ctgctcgtct?tccacgtgga?gggtcgctga?tctggaacca???60
gcggtgccga?tgcgcggcaa?agtgtcgaga?aattggcgta?ggggacgagg?tgcccgagga??120
aaccattttc?tgttttcagg?ggcgttcgaa?ttgggttgaa?tctgctcgtc?cttcgtcact??180
cgcatcattc?tacgcaaggg?agcggagaac?atttacctcg?catcagagtc?tggtggtgac??240
ccgaaggggg?atagtgtgag?ctaaatctca?aattatattc?attttcggta?attggaatga??300
agttttaaaa?cacaccacct?gtggccagca?taaataaggt?tacctttggt?tggcttaggg??360
ttcgcttagt?ttttatttat?tgatgatttt?tctacgtcta?tttgcgctgg?taggggggaa??420
gggattggac?acgggaatgg?aattagggaa?cacttgtgtt?gtctaaaggt?gaaagctaaa??480
tcaagcagga?ggtgacacca??????????????????????????????????????????????500
<210>2
<211>500
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
＜221〉promotor
<222>(1)..(500)
＜223〉promotor of NCgl1305
<400>2
ttttggcggg?cgcttcggcg?aaaatacatg?ggcctacaac?gccgtctaag?cgtgaaactg???60
ggggatgggg?atacctggaa?tcattcgggg?acgtgatgcg?gtggaggggt?ggtgcgggca??120
taatctgaca?gtgtgtccgt?tttcattttc?aaaaaatgca?ggtcggacat?attcaaaagt??180
attacctttt?tggtttgtct?gtattcagct?tgttttgggt?gggtttccgg?cttatcatga??240
tgggtgactt?accgcttaat?tggaaaaaag?tgtgatccac?cacaaatcta?ttgcggggga??300
gcctgggaaa?ctaggtaaaa?atttttgcca?aattgtgcaa?tcgttttcac?aacctgagaa??360
tgtcacaaca?cattaagtgg?taggcgctga?ggaatcgaat?ccgattcttt?ttcggcccaa??420
ttcgtaacgg?cgatcctctt?aagtggacaa?gaaagtctct?tgcccgcggg?agacagaccc??480
tacgtttaga?aaggtttgac??????????????????????????????????????????????500
<210>3
<211>33
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase primer 1 of promotor of NCgl1504 gene
<400>3
aggatatcgg?atccagcgga?gacatttacc?tcg????33
<210>4
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase primer 2 of promotor of NCgl1504 gene
<400>4
gagcatatgt?gtcacctcct?gcttga????????????26
<210>5
<211>33
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase primer 3 of promotor of NCgl1305 gene
<400>5
aggatatcgg?atccgtattc?agcttgtttt?ggg????33
<210>6
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase primer 4 of promotor of NCgl1305 gene
<400>6
gagcatatga?aacctttcta?aacgta????????????26
<210>7
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase primer 5 of IysC
<400>7
gacaggacaa?gcactggttg??????????????????20
<210>8
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase primer 6 of lysC
<400>8
aggatatcct?ttgtgcacct?ttcgatc????????27
<210>9
<211>28
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase primer 7 of lysC
<400>9
gatatcatat?ggccctggtc?gtacagaa????28
<210>10
<211>25
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase primer 8 of lysC
<400>10
ccagcctgag?agcccgtgaa?agatt????25
<210>11
<211>687
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
＜221〉gene
<222>(1)..(687)
<223>NCgl1504
<400>11
gtgggagatg?ttgtaaaagg?caacgacgcg?cacaccggag?acggtgatac?gcgccgaaaa?????60
attcttctca?tcctgttgga?acgtgcaccg?gtgatcgctt?cagatattgc?tgaacagctt????120
cagctttcaa?ctgtgggagt?gcgcaggcac?ctagacaact?tggttgaaga?aaatctggcg????180
gaggcggcaa?atccgcgcca?gaacccatat?gagcccaaaa?tgcgcggtag?gccagcaaaa????240
acttatcggc?ttactgataa?aggtcgctca?atcttcggcc?acgaatatga?ttcccttgct????300
gcggcagctc?tagccactct?tcgagaggtc?ggcggagatg?atgcagtaag?gcaatttgct????360
agaaagcgga?tcgaaacaat?tgttgagggt?attaccccag?cagatgtcac?agatcaatca????420
atcgaagata?cagccaaatc?tttagttgaa?gcttttagtc?ggcatggtta?tgcagcaact????480
gtcgatgcca?ctcgaaacgg?gttgcaactc?tgccagcatc?actgtccaat?atctacagtc????540
gccacggaat?ttccggaact?gtgtgaggca?gagcatcaag?cagtctcaga?acttttgggg????600
cagcacacgc?aaccattggc?aacaatcgcg?gacggccacg?gcatctgcac?aacaaatatt????660
ccattgacac?ccatcaaaca?ctcctga????????????????????????????????????????687
<210>12
<211>2052
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
＜221〉gene
<222>(1)..(2052)
<223>NCgl1305
<400>12
atggcgtcca?aactgacgac?gacatcgcaa?catattctgg?aaaaccttgg?tggaccagac????60
aatattactt?cgatgactca?ctgtgcgact?cgccttcgct?tccaagtgaa?ggatcaatcc???120
attgttgatc?aacaagaaat?tgactccgac?ccatcagttc?ttggcgtagt?accccaagga???180
tccaccggta?tgcaggtggt?gatgggtgga?tctgttgcaa?actattacca?agaaatcctc???240
aaacttgatg?gaatgaagca?cttcgccgac?ggtgaagcta?cagagagttc?atccaagaag???300
gaatacggcg?gagtccgtgg?caagtactcg?tggattgact?acgccttcga?gttcttgtct???360
gatactttcc?gaccaatcct?gtgggccctg?cttggtgcct?cactgattat?taccttgttg???420
gttcttgcgg?atactttcgg?tttgcaagac?ttccgcgctc?caatggatga?gcagcctgat???480
acttatgtat?tcctgcactc?catgtggcgc?tcggtcttct?acttcctgcc?aattatggtt???540
ggtgccaccg?cagctcgaaa?gctcggcgca?aacgagtgga?ttggtgcagc?tattccagcc???600
gcacttctta?ctccagaatt?cttggcactg?ggttctgccg?gcgataccgt?cacagtcttt???660
ggcctgccaa?tggttctgaa?tgactactcc?ggacaggtat?tcccaccgct?gattgcagca???720
attggtctgt?actgggtgga?aaagggactg?aagaagatca?tccctgaagc?agtccaaatg???780
gtgttcgtcc?cattcttctc?cctgctgatt?atgatcccag?cgaccgcatt?cctgcttgga???840
cctttcggca?tcggtgttgg?taacggaatt?tccaacctgc?ttgaagcgat?taacaacttc???900
agcccattta?ttctttccat?cgttatccca?ttgctctacc?cattcttggt?tccacttgga???960
ttgcactggc?cactaaacgc?catcatgatc?cagaacatca?acaccctggg?ttacgacttc??1020
attcagggac?caatgggtgc?ctggaacttc?gcctgcttcg?gcctggtcac?cggcgtgttc??1080
ttgctctcca?ttaaggaacg?aaacaaggcc?atgcgtcagg?tttccctggg?tggcatgttg??1140
gctggtttgc?tcggcggcat?ttccgagcct?tccctctacg?gtgttctgct?ccgattcaag??1200
aagacctact?tccgcctcct?gccgggttgt?ttggcaggcg?gtatcgtgat?gggcatcttc??1260
gacatcaagg?cgtacgcttt?cgtgttcacc?tccttgctta?ccatcccagc?aatggaccca??1320
tggttgggct?acaccattgg?tatcgcagtt?gcattcttcg?tttccatgtt?ccttgttctc??1380
gcactggact?accgttccaa?cgaagagcgc?gatgaggcac?gtgcaaaggt?tgctgctgac??1440
aagcaggcag?aagaagatct?gaaggcagaa?gctaatgcaa?ctcctgcagc?tccagtagct??1500
gctgcaggtg?cgggagccgg?tgcaggtgca?ggagccgctg?ctggcgctgc?aaccgccgtg??1560
gcagctaagc?cgaagctggc?cgctggggaa?gtagtggaca?ttgtttcccc?actcgaaggc??1620
aaggcaattc?cactttctga?agtacctgac?ccaatctttg?cagcaggcaa?gcttggacca??1680
ggcattgcaa?tccaaccaac?tggaaacacc?gttgttgctc?cagcagacgc?tactgtcatc??1740
cttgtccaga?aatctggaca?cgcagtggca?ttgcgcttag?atagcggagt?tgaaatcctt??1800
gtccacgttg?gattggacac?cgtgcaattg?ggcggcgaag?gcttcaccgt?tcacgttgag??1860
cgcaggcagc?aagtcaaggc?gggggatcca?ctgatcactt?ttgacgctga?cttcattcga??1920
tccaaggatc?tacctttgat?caccccagtt?gtggtgtcta?acgccgcgaa?attcggtgaa??1980
attgaaggta?ttcctgcaga?tcaggcaaat?tcttccacga?ctgtgatcaa?ggtcaacggc??2040
aagaacgagt?aa??????????????????????????????????????????????????????2052
<210>13
<211>1500
<212>DNA
＜213〉Corynebacterium glutamicum
<220>
＜221〉gene
<222>(1)..(1500)
＜223〉217: initial 1490: stop (lysC gene)
<400>13
tcgcgaagta?gcacctgtca?cttttgtctc?aaatattaaa?tcgaatatca?atatatggtc???60
tgtttattgg?aacgcgtccc?agtggctgag?acgcatccgc?taaagcccca?ggaaccctgt??120
gcagaaagaa?aacactcctc?tggctaggta?gacacagttt?ataaaggtag?agttgagcgg??180
gtaactgtca?gcacgtagat?cgaaaggtgc?acaaaggtgg?ccctggtcgt?acagaaatat??240
ggcggttcct?cgcttgagag?tgcggaacgc?attagaaacg?tcgctgaacg?gatcgttgcc??300
accaagaagg?ctggaaatga?tgtcgtggtt?gtcgtctccg?caatgggaga?caccacggat??360
gaacttctag?aacttgcagc?ggcagtgaat?cccgttccgc?cagctcgtga?aatggatatg??420
ctcctgactg?ctggtgagcg?tatttctaac?gctctcgtcg?ccatggctat?tgagtccctt??480
ggcgcagaag?cccaatcttt?cacgggctct?caggctggtg?tgctcaccac?cgagcgccac??540
ggaaacgcac?gcattgttga?tgtcactcca?ggtcgtgtgc?gtgaagcact?cgatgagggc??600
aagatctgca?ttgttgctgg?tttccagggt?gttaataaag?aaacccgcga?tgtcaccacg??660
ttgggtcgtg?gtggttctga?caccactgca?gttgcgttgg?cagctgcttt?gaacgctgat??720
gtgtgtgaga?tttactcgga?cgttgacggt?gtgtataccg?ctgacccgcg?catcgttcct??780
aatgcacaga?agctggaaaa?gctcagcttc?gaagaaatgc?tggaacttgc?tgctgttggc??840
tccaagattt?tggtgctgcg?cagtgttgaa?tacgctcgtg?cattcaatgt?gccacttcgc??900
gtacgctcgt?cttatagtaa?tgatcccggc?actttgattg?ccggctctat?ggaggatatt??960
cctgtggaag?aagcagtcct?taccggtgtc?gcaaccgaca?agtccgaagc?caaagtaacc?1020
gttctgggta?tttccgataa?gccaggcgag?gctgcgaagg?ttttccgtgc?gttggctgat?1080
gcagaaatca?acattgacat?ggttctgcag?aacgtctctt?ctgtagaaga?cggcaccacc?1140
gacatcacct?tcacctgccc?tcgttccgac?ggccgccgcg?cgatggagat?cttgaagaag?1200
cttcaggttc?agggcaactg?gaccaatgtg?ctttacgacg?accaggtcgg?caaagtctcc?1260
ctcgtgggtg?ctggcatgaa?gtctcaccca?ggtgttaccg?cagagttcat?ggaagctctg?1320
cgcgatgtca?acgtgaacat?cgaattgatt?tccacctctg?agattcgtat?ttccgtgctg?1380
atccgtgaag?atgatctgga?tgctgctgca?cgtgcattgc?atgagcagtt?ccagctgggc?1440
ggcgaagacg?aagccgtcgt?ttatgcaggc?accggacgct?aaagttttaa?aggagtagtt?1500
1500
Claims (8)
1. promoter nucleic acid molecule, it has the nucleotide sequence of SEQ ID NO:1 or 2.
2. recombinant vectors, it comprises the promotor of the claim 1 that is connected with target gene encoding sequence operability.
3. host cell, its recombinant vectors with claim 2 transforms.
4. the host cell of claim 3, it belongs to excellent bacillus (Corynebacterium) and belongs to.
5. the host cell of claim 4, it is Corynebacterium glutamicum (Corynebacterium glutamicum) KCCM 10831 that is transformed by recombinant vectors, and described recombinant vectors comprises the promotor of the called after SEQ ID NO:1 that is connected with lysC gene coded sequence operability.
6. the host cell of claim 4, it is Corynebacterium glutamicum (Corynebacterium glutamicum) KCCM 10830 that is transformed by recombinant vectors, and described recombinant vectors comprises the promotor of the called after SEQ ID NO:2 that is connected with lysC gene coded sequence operability.
7. express the method for target gene, described method comprises that cultivation is according to each host cell among the claim 3-6.
8. the method for claim 7, wherein said target gene is the lysC gene.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2007-0004386 | 2007-01-15 | ||
| KR1020070004386A KR100789274B1 (en) | 2007-01-15 | 2007-01-15 | Novel promoter nucleic acid molecule derived from Corynebacterium glutamicum recombinant vector comprising the promoter host cell comprising the recombinant vector and method of expressing a gene using the host cell |
| KR1020070004386 | 2007-01-15 | ||
| PCT/KR2008/000246 WO2008088158A1 (en) | 2007-01-15 | 2008-01-15 | Novel promoter nucleic acid molecule derived from corynebacterium glutamicum, recombinant vector comprising the promoter, host cell comprising the recombinant vector and method of expressing gene using the host cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101605890A true CN101605890A (en) | 2009-12-16 |
| CN101605890B CN101605890B (en) | 2013-05-01 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008800018883A Active CN101605890B (en) | 2007-01-15 | 2008-01-15 | Novel promoter nucleic acid molecule derived from corynebacterium glutamicum, recombinant vector comprising the promoter, host cell comprising the recombinant vector and method of expressing gene usin |
Country Status (7)
| Country | Link |
|---|---|
| US (1) | US8426195B2 (en) |
| JP (1) | JP5266252B2 (en) |
| KR (1) | KR100789274B1 (en) |
| CN (1) | CN101605890B (en) |
| BR (1) | BRPI0806252B8 (en) |
| DE (1) | DE112008000181T9 (en) |
| WO (1) | WO2008088158A1 (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191247A (en) * | 2010-03-05 | 2011-09-21 | Cj第一制糖株式会社 | Enhanced promoter and method for producing L-lysine usinge the same |
| CN108350464A (en) * | 2015-12-07 | 2018-07-31 | 齐默尔根公司 | Promoter from corynebacterium glutamicum |
| CN110869504A (en) * | 2017-06-07 | 2020-03-06 | 齐默尔根公司 | Promoter from C.glutamicum and use thereof for regulating the expression of auxiliary genes |
| CN111670252A (en) * | 2018-03-20 | 2020-09-15 | Cj第一制糖株式会社 | Novel promoter and application thereof |
| US11293029B2 (en) | 2015-12-07 | 2022-04-05 | Zymergen Inc. | Promoters from Corynebacterium glutamicum |
| WO2022152036A1 (en) * | 2021-01-12 | 2022-07-21 | 中国科学院天津工业生物技术研究所 | Polynucleotide having promoter activity and use thereof in production of traget compounds |
| CN115322989A (en) * | 2021-05-10 | 2022-11-11 | 中国科学院天津工业生物技术研究所 | Polynucleotide having promoter activity, use thereof, and method for producing target compound |
| CN115322989B (en) * | 2021-05-10 | 2024-09-27 | 中国科学院天津工业生物技术研究所 | Polynucleotide having promoter activity, use thereof and method for producing target compound |
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| KR101072708B1 (en) * | 2008-12-17 | 2011-10-11 | 씨제이제일제당 (주) | A corynebacteria having enhanced 5'-xanthosine monophosphate productivity and a method of producing 5'-xanthosine monophosphate using the same |
| KR101072720B1 (en) * | 2008-12-17 | 2011-10-11 | 씨제이제일제당 (주) | A corynebacteria having enhanced 5'-guanosine monophosphate productivity and a method of producing 5'-guanosine monophosphate using the same |
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| CN103153044B (en) | 2010-10-08 | 2015-06-17 | 韩国生命工学研究院 | Latex secretory tissue-specific srpp promoter derived from hevea brasiliensis and use thereof |
| US8846367B2 (en) * | 2010-11-18 | 2014-09-30 | Green Phenol Development Co., Ltd. | Coryneform bacterium transformant and process for producing phenol using the same |
| KR20140066553A (en) | 2012-11-23 | 2014-06-02 | 삼성전자주식회사 | Novel promoter of corynebacterium genus |
| KR20140095850A (en) | 2013-01-25 | 2014-08-04 | 삼성전자주식회사 | Shuttle vector for Escherichia coli and Corynebacteria |
| KR101677368B1 (en) | 2015-04-02 | 2016-11-18 | 씨제이제일제당 (주) | A novel promoter and use thereof |
| KR101925930B1 (en) * | 2017-07-24 | 2019-02-26 | 씨제이제일제당 주식회사 | Microorganisms producing L-lysine and process for producing L-lysine using the same |
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|---|---|---|---|---|
| DE19931314A1 (en) * | 1999-07-07 | 2001-01-11 | Degussa | L-lysine producing coryneform bacteria and method for producing lysine |
| JP4623825B2 (en) * | 1999-12-16 | 2011-02-02 | 協和発酵バイオ株式会社 | Novel polynucleotide |
| JP2003265177A (en) * | 2002-03-13 | 2003-09-24 | Gekkeikan Sake Co Ltd | High protein expression system |
| WO2005021765A2 (en) | 2003-08-27 | 2005-03-10 | Orf Liftaekni Hf. | Enhancing accumulation of heterologous polypeptides in plant seeds through targeted suppression of endogenous storage proteins |
| DE102004035065A1 (en) * | 2004-07-20 | 2006-02-16 | Basf Ag | P-ET-TS expression units |
| KR100620092B1 (en) | 2004-12-16 | 2006-09-08 | 씨제이 주식회사 | Novel promoter nucleic acid derived from corynebacterium genus bacteria expression cassette comprising the promoter and vector comprising the cassette host cell comprising the vector and method for expressing a gene using the cell |
| JP2006214933A (en) | 2005-02-04 | 2006-08-17 | Seiko Epson Corp | Positioning system, terminal device, control method and program of terminal device, and computer readable recording medium having recorded control program of terminal device |
| JP4700395B2 (en) * | 2005-04-13 | 2011-06-15 | 株式会社豊田中央研究所 | Promoters for use under acidic conditions and use thereof |
| AU2007295159B2 (en) | 2006-09-15 | 2012-09-13 | Cj Cheiljedang Corporation | A Corynebacteria having enhanced L-lysine productivity and a method of producing L-lysine using the same |
-
2007
- 2007-01-15 KR KR1020070004386A patent/KR100789274B1/en active IP Right Grant
-
2008
- 2008-01-15 WO PCT/KR2008/000246 patent/WO2008088158A1/en active Application Filing
- 2008-01-15 US US12/523,272 patent/US8426195B2/en active Active
- 2008-01-15 BR BRPI0806252A patent/BRPI0806252B8/en active IP Right Grant
- 2008-01-15 JP JP2009545497A patent/JP5266252B2/en active Active
- 2008-01-15 DE DE200811000181 patent/DE112008000181T9/en not_active Ceased
- 2008-01-15 CN CN2008800018883A patent/CN101605890B/en active Active
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191247A (en) * | 2010-03-05 | 2011-09-21 | Cj第一制糖株式会社 | Enhanced promoter and method for producing L-lysine usinge the same |
| CN102191247B (en) * | 2010-03-05 | 2013-04-24 | Cj第一制糖株式会社 | Enhanced promoter and method for producing L-lysine usinge the same |
| CN108350464A (en) * | 2015-12-07 | 2018-07-31 | 齐默尔根公司 | Promoter from corynebacterium glutamicum |
| US11293029B2 (en) | 2015-12-07 | 2022-04-05 | Zymergen Inc. | Promoters from Corynebacterium glutamicum |
| CN110869504A (en) * | 2017-06-07 | 2020-03-06 | 齐默尔根公司 | Promoter from C.glutamicum and use thereof for regulating the expression of auxiliary genes |
| CN111670252A (en) * | 2018-03-20 | 2020-09-15 | Cj第一制糖株式会社 | Novel promoter and application thereof |
| CN111670252B (en) * | 2018-03-20 | 2023-12-12 | Cj第一制糖株式会社 | Novel promoter and application thereof |
| WO2022152036A1 (en) * | 2021-01-12 | 2022-07-21 | 中国科学院天津工业生物技术研究所 | Polynucleotide having promoter activity and use thereof in production of traget compounds |
| CN115322989A (en) * | 2021-05-10 | 2022-11-11 | 中国科学院天津工业生物技术研究所 | Polynucleotide having promoter activity, use thereof, and method for producing target compound |
| CN115322989B (en) * | 2021-05-10 | 2024-09-27 | 中国科学院天津工业生物技术研究所 | Polynucleotide having promoter activity, use thereof and method for producing target compound |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101605890B (en) | 2013-05-01 |
| BRPI0806252B8 (en) | 2017-05-02 |
| BRPI0806252A2 (en) | 2011-08-30 |
| US20100261256A1 (en) | 2010-10-14 |
| KR100789274B1 (en) | 2008-01-02 |
| DE112008000181T5 (en) | 2009-12-10 |
| JP2010515453A (en) | 2010-05-13 |
| WO2008088158A1 (en) | 2008-07-24 |
| JP5266252B2 (en) | 2013-08-21 |
| DE112008000181T9 (en) | 2010-04-08 |
| US8426195B2 (en) | 2013-04-23 |
| BRPI0806252B1 (en) | 2016-11-29 |
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